PrOpCom Making Nigerian Agricultural Markets Work for the Poor

Monograph Series # 26

DEFINITION OF OFADA RICE QUALITIES THROUGH VARIETAL IDENTIFICATION AND TESTING

By

National Cereals Research Institute (NCRI) Badeggi P.M.B. 8, Bida, Niger State, (J. C. Anounye, N. Danbaba, A.S. Gana and M. E. Abo)

And

Africa Rice Centre, (WARDA), c/o International Institute of Tropical Agriculture. PMB 5320, Oyo Road, Ibadan, Nigeria (G. Gregorio, O.A. Oladimeji, B. Athanson, O. Ajayi, and F.E. Nwilene)

August, 2007

Funding for this programme is provided by the United Kingdom’s Department for International Development (DFID)

40 Mississippi Street, Maitama, Abuja, Nigeria • Tel: +(234) 9 413 8291/2 • Fax: +(234) 9 413 829

1 Disclaimer

The PrOpCom Monograph Series seeks to provide a broader dissemination of the information and views collected through the efforts of the various service providers and consultants undertaking work in support of the PrOpCom programme. We believe this information will be useful in informing the policy dialogue in Nigeria and for improving the planning and execution of agricultural and agribusiness activities within the country.

The documents in this series represent the final deliverables as presented by the engaged service providers or consultants in responds to terms of reference of contracts let by PrOpCom in execution of its programme. They have not been further edited or editorially polished. Consequently, there is wide variation in the editorial quality of these documents. Nevertheless, the information contained in these document is deems useful for policy and planning purposes.

The views and opinions expressed in these documents are solely those of the authors of the document and do not necessarily represent the views of PrOpCom, SAII Associates, Chemonics International or PrOpCom’s funding agent, the Government of the United Kingdom’s Department for International Development (DFID)

Information from these documents can be freely used and quoted on condition that it is properly sourced to the concerned document.

2 PrOpCom Project: Services Agreement – OFADA-07

DEFINITION OF OFADA RICE QUALITIES THROUGH VARIETAL IDENTIFICATION AND TESTING

Submitted to

PrOpCom (Promoting Pro-Poor Opportunities Through Commodity and Service Markets) Plot 40, Mississippi - Street, Maitama Abuja, Nigeria

By

National Cereals Research Institute (NCRI) Badeggi P.M.B. 8, Bida, Niger State, Nigeria (J. C. Anounye, N. Danbaba, A.S. Gana and M. E. Abo)

And

Africa Rice Centre, (WARDA), c/o International Institute of Tropical Agriculture. PMB 5320, Oyo Road, Ibadan, Nigeria (G. Gregorio, O.A. Oladimeji, B. Athanson, O. Ajayi, and F.E. Nwilene)

3 Executive summary

‘Ofada’ is a generic name used to describe all rice produced and processed in the rice producing clusters of the South-West Nigeria. It has recently gained prominence and is fast gaining international attention. In this part of Nigeria, every rice seed planted and processed locally is taken to be ‘Ofada’ and this has been adulterated and misrepresented. Therefore, it has become necessary to identify and classify ‘Ofada’ rice through seed variety identification. The broad objective of this study was to identify and classify ‘Ofada’ rice from the clusters where they are grown. Specific objectives include (a) identify variety characteristics and properties of available ‘Ofada’ rice seeds presently with farmers (b) carry out physical and chemical analysis, morphological and molecular characterization to establish the true identity of ‘Ofada’ rice (c) establish seed variety with the correct genotype and physical properties of the ‘Ofada’ rice. Samples were collected from rice growing communities of Ogun, Osun, Ekiti and states. The physical and chemical characteristics were determined using the Standard Evaluation Systems (SES). The grains were broadly grouped into two-variety types based on the hull and kernel color. Grain physical dimension also classified the grains into medium to long grain types and this was substantiated by the sieve analysis. There was a high milling return, but suspected impurities and contaminants gave rise to the low head rice count. The differences in grain hull and kernel weight, particle sizes etc arose from this contamination and not from genetic differences. The results for the cooking characteristics showed that the samples had high peak and setback viscosities, which were expected. The results also indicated that, like all other cereals, starch consistency would be low and subject to retro gradation on standing. The high peak viscosity was indicative of high amylose content. Any differences in pasting properties were not due to particle size differences, but variety mixtures within the samples. Results of the study showed that the ‘Ofada’ rice varieties have similar amino acid profiles and related very closely to two of the checks

4 FARO 46 (ITA 150) and FARO 11(OS 6). From the physical and chemical analysis therefore, it can be concluded that ‘Ofada’ rice is some released variety that has been contaminated with other varieties all over the years from the farmers’ field. However, there was the red rice ( Steud.) in most of the ‘Ofada’ rice seed samples. Further studies on morphological and molecular characterization of Twenty six rice genotypes, including 12 Ofada rice collected by PrOpCom from farmers’ fields and 14 NERICA/ITA genotypes from WARDA (NERICA 1, NERICA 3, NERICA 5, WAB450-1-B-P-180-HB, WAB189-B-B-B-8-HB, WAB33-25, WAB450-24-3-2-P18-HB, ITA 150, ITA 301, ITA 117, ITA 321, OS 6 and WAB706-3-4-K4-KB) were physically characterized to provide some information on grain type and purity of the seeds. The grain size, shape and colour, awn, pubescence, hull colour, etc. were observed and documented. They were seeded on 23 February 2007 in a wet bed at field F of the International Institute of Tropical Agriculture (IITA), Ibadan farm and transplanted in the Lowland Field on 14 March 2007 for morphological characterization at the vegetative and reproductive stages. Molecular characterization was conducted through DNA isolation, primer testing, PCR amplification, fingerprinting, and genetic similarity and cluster analysis. The Ofada rice collection showed mixtures, as demonstrated in the physical characteristics of the grain. There was significant variation in the grain size of Ofada rice, which ranged from medium or intermediate to very long grains. The kernels were red and white, the red kernel types varying from red to deep red. Based on the dendrogram the following comparisons can be made: OS 6 and WAB33-25 are the same and are in the same group as Ofada 10, WAB450-1-B-P, WAB189-B-B-B and ITA321; ITA150 is close to Ofada 6,7,8 and 9; Ofada 1-5 belong to one group distinct from all the other varieties; while Ofada 11 and 12 are closely related to WAB450-24, WAB706-3-4-K, ITA117 and ITA301. For seed purification, 10 individual plants per Ofada collection per replication were harvested. These plants selected will be planted side by side in the 2007 rainy season. Based on specific criteria (grain color, size and shape,

5 height, etc.), the rows matching the Ofada characteristics will be selected for seed multiplication and further morphological characterization and molecular profiling to assure purity and establish proper identity. The variability within and between samples explains the complexity in identifying the real Ofada. The information generated from the molecular characterization would have been more reliable if the seed was not mixed. Molecular characterization therefore should be repeated after the Ofada rice accessions have been purified and isolated. It is therefore necessary in the interim to purify the ofada collections to address the issue of adulteration; seed mixtures and the resultant low yield on farmers’ fields as follows (1) purify Ofada 1-5 and 12 for farmers in Ogun and Lagos States, (2) Purify Ofada 6-11 and 12 for farmers in Osun, Ekiti and Lagos States, (3) Source for fresh foundation seeds of ITA 150 (FARO 46) and OS 6 (FARO 11) already released as varieties in Nigeria from National Seed Service (NSS) in collaboration with NCRI and distributed to farmers for cultivation in the Northwest rice growing clusters in Nigeria.

6 1.0 Introduction:

Rice is one of the most important crops in Nigeria and its production represents a significant part of the government strategy to overcome food shortage and improve self-sufficiency for both local consumption and export. Presently, annual per capita consumption of milled rice stands at 25kg per person. There are two cultivated species of rice, the Oryza glaberrima Steud and the O. sativa L. Following the introduction of O. sativa from Asia, the indigenous rice species O. glaberrima Steud was pushed to the marginal areas. Early released varieties were selected from local and introduced materials. One of the earliest released varieties was the upland variety FARO 3 (Agbede), which was selected from a complex collection of upland varieties believed to have been introduced by veterans returning from the First World War. As breeding effort continued, OS 6 was introduced from Zaire (Democratic Republic of Congo) and released as FARO 11 to replace Agbede. Additional varieties such as ITA 150 and NERICA 1 have been released as FARO 46 and FARO 55 and are in cultivation in the South West region of Nigeria. ‘Ofada’ therefore, is a generic name used to describe any kind of rice cultivated and processed in a group of communities in Ogun state and some rice producing clusters in South West Nigeria. They are grown under the upland ecosystem where the fields are not bonded but are prepared and seeded under dry conditions and depend on rainfall for moisture. Ofada rice is a special eaten occasionally by natives of this region and traditionally served in Tomatococcus danielli leaves. Two kinds of ‘Ofada’ rice are identified by farmers and consumers of the rice based on the time of harvest, color of the endosperm and intensity of flavor. They are: “white Ofada” and “brown or red ‘Ofada’. Cooked Ofada rice is usually eaten with a special kind of sauce prepared using pepper (‘Atarodo,’ Tatase’), onion, locust beans, palm oil and assorted meat. The mode of preparation, serving and emotional value obtained in eating ‘Ofada’ rice is traced to its indigenousness that connects people to their cultural heritage and food habit.

7 The morphology of rice is divided into the vegetative phase (including germination, seedling, and tillering stages) and the reproductive phase (including panicle initiation and heading stages).

The appearance of milled rice is important to consumers. The physical dimensions of rice kernels are of vital interest to those engaged in many facets of the rice industry. Thus grain size and shape are the first criteria of rice quality to be considered in releasing varieties for commercial production. Rice varieties may be classified into grain-type categories based upon two physical parameters, length and shape. Length is a measure of the rice kernel in its greatest dimension. The shape is determined by the length-width ratio. Although grain size and shape can be visually classified, more exact measurements are needed for classification for critical comparison of varieties.

‘Ofada’ rice has recently gained prominence and is fast gaining international attention. In an apparent move to respond to this and other factors like consumer demand, brand identity, improved production, processing and marketing of this specialty rice as a means of improving the living standard of the people involved in the ‘Ofada rice value chain’, PrOpCom funded research on quality evaluation of ‘Ofada’ rice produced in South-Western states of Nigeria. This is aimed as giving answer to the challenges of adulteration, misrepresentation and low productivity. Rice grain quality has become very important among Nigerians, as quality preferences vary among ethnic groups and the preparation to which rice will be subjected. Therefore, quality identification of the local rice produced and consumed in Nigeria is the basis for the establishment of brands for various varieties planted and consumed as local rice. Molecular markers have proven to be very useful for varietal identification and DNA fingerprinting in several rice cultivars (Virk et al., 2000; Ren et al., 2003; Garris et al., 2005). Simple sequence repeats (SSR) are tandemly repeated

8 sequence motifs that are ubiquitously distributed throughout the eukaryotic genome and are valued because they are genetically defined, highly variable, co dominant markers, and because of their ease of detection by PCR amplification of small amounts of DNA.

1.2 Rationale for the study Rice, unlike most other cereals, is consumed as a whole grain. Therefore, quality characteristics and general appearance of a given rice variety is of utmost importance. Grain quality in rice, therefore depends on the intended end-use. For the rice farmers, rice is a source of income and livelihood and therefore a high yielding variety is required. The rice miller bases his quality upon total recovery and the proportion of broken and head rice on milling. Consumer and homemakers base their concept of quality on the grain physical appearance, size and shape, the behavior upon cooking and taste, tenderness and in rare cases flavor of cooked rice. This makes branding in rice a difficult task. If quality characteristics of rice as defined by all the interest groups are well understood, branding of the given variety will not be difficult. In dealing with the issue of misrepresentation and clear brand identification of ‘Ofada’ rice, PrOpCom facilitated this project for the purpose of identification and variety testing of some ‘Ofada’ rice varieties as a means of establishing quality standards for ‘Ofada’ rice. This information is intended to be documented, communicated and be used for improvement/ transformation of selected rice varieties. It is also believed that standards obtained from this research when accepted and used by the rice industries, will be very important elements for the establishment of brand identity, and are also useful for market development. Commercial end-users and industrial buyers have expressed a strong need for consistent ‘Ofada’ rice quality as a basis for continuous patronage and inclusion of the product in their served menus. More over, PrOpCom sees this effort as having pro-poor benefits for farmers

9 as identification of variety characteristics is a basis for determining which ‘Ofada’ rice seed variety is selected and improved and made available to farmers as part of the ‘Ofada Rice Value Chain’ Development Action Plan. 1.3 Scope of the Study In a general consensus from stakeholders at a planning meeting, establishment of a programme to improve the ‘Ofada Rice Value Chain’ Development must begin with identification, through farmer evaluation and laboratory analysis. Consequently, PrOpCom has commissioned the National Cereals Research Institute (NCRI) (hereafter referred to as Service Provider) to undertake variety identification and testing of collected ‘Ofada’ rice seed samples. The tasks include: 1. Identifying variety characteristics and properties of available seeds of ‘Ofada’ rice presently planted by farmers in rice production clusters of South Western Nigeria. Undertake laboratory analysis to identify and select seeds whose properties meet the required properties of ‘Ofada’ rice quality; 2. Establish seed varieties with the correct genotype (through molecular and morphological characterization) and physical properties of the true ‘Ofada’ rice. 1.4 Specific Objectives and Deliverables Service providers are expected to complete the following deliverables. Identify variety characteristics and properties of available ‘Ofada’ rice seeds presently planted by farmers in the rice producing clusters of South Western Nigeria. 1. Use laboratory analysis, morphological and molecular characterization to identify and select seeds whose properties meet the required properties of Ofada rice 2. Undertook laboratory analysis to identify and select seeds whose properties meet the required properties of ‘Ofada’ rice quality;

10 2.0 Methodology 2.1 Materials 2.1.1 Plant Materials PrOpCom supplied Twelve (12) ‘Ofada’ rice samples while WARDA (Ibadan) supplied fourteen (14) rice samples for the study. The ‘Ofada’ rice samples were collected from Ogun, Osun, Ekiti, and Lagos states. The list of the Ofada rice collections, sources and location is presented in Table 1.

Table 1. List of Ofada rice seeds collected by PrOpCom and their sources and location

S/No (Ref. Preferrerd listing) Variety Source(s) Location Name Ofada 1 Ofada white Madam Abebi, J. Suin Obafemi/Owode LGA, Ogun state Ofada 2 Ofada red Madam Abebi J. Suin Obafemi/Owode LGA, Ogun State Ofada 3 Ofada, Ewekoro 1 Mr. O. Fadero Ewekoro Ewekoro LGA, Ogun State Ofada 4 Ofada, Ewekoro 2 Mr. Isaika K. Ewekoro Ewekoro LGA, Ogun State Ofada 5 Ofada Pr. Sikiru Popoola Lufoko Village, via Ayiwere, Obafemi/ Owode LGA, Ogun State Ofada 6 Ofada red Mrs. Eunice Adefioye Orin Oke, Osun State Ofada 7 Ofada Canada Mrs. Eunice Adefioye Earin Oke, Osun State Ofada 8 Ofada red Pastor Adefioyo Erin Oke, Osun State Ofada 9 Igbemo red Mr. Ajayi Abiodun Igbemo, Ekiti State Ofada 10 Igbemo white Mr. Ajayi Abiodum Igbemo, Ekiti State Ofada 11 Ofada while Mrs. Alice Adunni Ero, Ekiti State Ofada 12 Ofada Chief Balogun Olatunji Epe LGA, Lagos State

The 14 rice samples jointly added by WARDA (Ibadan) and NCRI are: NERICA 1, NERICA 3, NERICA 5, NERICA 8, WAB 450-1-B-P-180-HB, WAB 189-B-B-B-8-HB, WAB33-25, WAB 450-24-3-2-P18-HB, ITA 150, ITA 301, ITA 117, ITA 321, OS 6 and WAB 706-3-4-K4-KB. Out of these varieties, seven of them have been officially released in Nigeria through NCRI for commercial cultivation. These include NERICA 1 (FARO 55), WAB 189-B-B-B-8-HB (FARO 54), ITA 150 (FARO 46), ITA 301 (FARO 48), ITA 117 (FARO 47), ITA 321 (FARO 53) and OS 6 (FARO 11). They have been subjected to similar tests as

11 the ‘Ofada’ rice varieties as part of PrOpCom’s project on improving ‘Ofada Rice Value Chain’.

2.2 Methods 2.2.1 Physical and Chemical Characterizations

All the rice samples were subjected to physical and chemical characterization, milling, and cooking tests using the Standard Evaluation System (SES) for rice (IRRI 1996).

Grain Length, Width and Shape Twenty rice grain kernels were measured for grain length and width, while the length determined the shape: width ratio. The mean figures were obtained and grouped according to standard.

Weight of 1000 whole grain Twenty grams of brown rice was obtained after removing grains of other crops, foreign matter (anything other than rice grain-rachis, branches, chaff or empty paddy, grain less than ¼ of the length of complete grain, sand earth, straws). The weight of 1000 grains was then calculated using the equation below. One Thousand-grain weight = 1000 x 20/number of grains in the 20 g

Milling Quality Duplicate of 125 g rough rice samples were used for milling determinations. Rough rice samples were dehulled with a Satake laboratory sheller. Brown rice was milled with McGill number 2 for 30 seconds. Whole grains (head rice) were separated from the total milled rice. The % head rice and hull were determined as follows: Head rice (%) = weight of total milled rice Weight of rough rice x 100 Hull (%) = Weight of hull  Weight of rough rice x 100

12 Moisture Content Moisture content was determined using the Air-Oven method. Samples were placed in a separate aluminum moisture dish and heated in the oven to constant weight. The weight differences before and after heating determined moisture content. The formula is presented below:

Moisture Content (%) = (W1 – W2)  (W1 – W0) x 100 Where

W0 = Constant weight of moisture dish

W1= Weight of dish + sample before drying

W2 = Weight of dish + sample after drying

Protein Content Protein content was determined using the micro Kjeldahl method. A nitrogen- protein conversion factor of 5.95 was used. Gelatinization Temperature Duplicate sets of six whole kernels were selected and placed in a plastic container (2” x 2” x 1”) and 10ml of 1.7% potassium hydroxide (KOH) solution was added. The containers were covered and incubated for 23 hr at room temperature (30  20C). The endosperm was rated visually based on a 7-point numerical spreading value.

Grain Elongation Twenty whole grains were soaked in 20 ml of distilled water for 30 min., before placing them in a water bath where temperature was maintained at 900C for 12 minutes. Cooked whole grains were selected and measured. Grain elongation was estimated as the ratio of the average length of cooked rice grain to average length of raw rice grains.

Water Uptake Ratio (WUR) Eight grams of rice was cooked in excess water. The cooked rice was weighed and Water Uptake Ratio (WUR) calculated as follows: WUR = Weight of cooked rice  Weight of uncooked rice.

13 Grain Particle Size Analysis The grain particle size was analyzed at the Department of Civil Engineering, University of Agriculture, Makurdi using the Universal Sieve Shakers (Endicott Ltd London, England). The sieves were arranged in descending aperture size order (i.e. 3.35, 2.33, 1.70, 1.18 and 0.85mm). The shakers were then operated for 5 minutes after which the quantity of grain retained in each sieve was recorded.

Amino Acid Analysis The Amino Acid Profile (AAP) was evaluated at the Zoology Department of the University of Jos using Sequential Multi-analyzer Technicon (TSM-1 Model DNA 0209). The samples were dried to constant weight, defatted, hydrolyzed, evaporated in a rotary evaporator and then loaded into the analyzer. The amounts loaded were between 5-10 micro liters. This was dispensed into the cartridge of the analyzer. The samples were separated and analyzed as free acidic and basic amino acids. The period of analysis lasted for 76 minutes. Amino acid content calculated as g/100g protein was computed by measuring the net height of each peak produced by the chart recorder (Figures 1-15). The half heights of the peaks were determined and the width of the peak at half height was accurately measured. Approximate area of each peak was then obtained by multiplying the height with the width at half-height. With the internal amino acid standard and constant factors, the amino acid content was calculated as:

NH x NH  2 x Sstd x C = D x 16  SW x N2%x10xVLxNHxW

(Nileu)

Where:

NH = Net height of peaks, W = Width of peaks, D = Dilution factor, Sstd = Standard, C= Constant, SW = Sample weight, VL = Volume loaded.

Pasting Characteristics

14 Pasting profile of the samples was evaluated using the 20 minutes-Rapid Visco-Analyzer (RVA) (Newport scientific 910140, Sydney Australia). Rice samples were milled into flour and 3 g of the sample was mixed with 25 ml of distilled water. The disposable plastic stirring paddle was placed in the cup and rotated by hand for 15-30 seconds to wet the samples. The sample cup and paddle were inserted into the RVA and the test cycle began. Sample temperature was equilibrated at 500C for 2 minutes, and the sample was then put on a heating cycle for 8 minutes with a minimum temperature of 500C. The viscosity profiles were recorded on the portable computer attached to the instrument.

2.2.2 Morphological and Molecular Characterizations Twenty-six rice genotypes, including 12 Ofada rice from farmer’s fields (Table 2) and 14 NERICA/ITA genotypes from WARDA (Table 1) were seeded on 23 February 2007 in a wet bed at field F of the IITA-Ibadan farm (Figure 1). Seedlings were transplanted in the Lowland Field on 14 March 2007 for morphological characterization at the vegetative and reproductive stages. All the 26 genotypes were planted in three replications with 3 rows and 46 hills per row (Figure 2). The 14 NERICA/ITA genotypes from WARDA are: NERICA 1, NERICA 3, NERICA 5, WAB450-1-B-P-180-HB, WAB189-B-B-B-8-HB, WAB33-25, WAB450-24-3-2-P18-HB, ITA 150, ITA 301, ITA 117, ITA 321, OS 6 and WAB706-3- 4-K4-KB.

15 Figure 1. Germination of Ofada rice seeded in wet bed 9 days before transplanting at IITA farm Ibadan

Figure 2. Transplanted Ofada rice in the lowland field 9 days after transplanting at IITA farm Ibadan

Measurement of morphological characteristics The morphology of rice is divided into the vegetative phase (including germination, seedling, and tillering stages) and reproductive phase (including panicle initiation and heading stages). Figure 3 shows the field stand of the crop as at 29th May 2007. Specific characters were observed at different growth stages using codes according to the IRRI Standard Evaluation Systems, July 1996 (SES IRRI 1996) (Table 2).

16 Figure 3. Ofada rice field performance 93 days after seeding at WARDA lowland plot at IITA farm, Ibadan

Molecular characterization A total of 34 SSR primers were available for the study. Each primer had a different Tm value i.e. melting temperature based on the sequences and this determines the annealing temperature. All PCR reactions were performed on a PTC-200 Peltier Thermal Cycler (MJ Research, USA), in a 6.6ul reaction containing 10ng of genomic DNA; 25mM MgCl2; 1mM dNTP mix, 5uM of each Primer, 5 units of Taq polymerase; and 10X Buffer.

DNA Isolation Leaf tissue of each genotype was collected from the field and placed on ice for transport to the laboratory. Total genomic DNA was isolated from fresh leaf tissues using the modified Dellaporta method of DNA extraction (http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb2//r03-032.pdf, Dellaporta et al., 1983): 0.25g of leaf tissue was ground to powder in a 2ml eppendorf tube using liquid nitrogen after which pre-heated extraction buffer (1M Tris pH8, 0.5M EDTA, 5M NaCl at 650C) was added.

17 Table 2: Specific characters measured at different growth stages Characters Growth Stage Seedling height (SH) 2-3 (5 leaf stage) Leaf length (LL) 6 Leaf width (LW) 6 Leaf pubescence (LBP) 5-6 Leaf blade color (LBC) 4-6 Basal Leaf Leaf blade color (BLSC) 3-5 Leaf angle (LA) 4-5 Flag leave angle (FLA) 5 Ligule length (LgL) 5 Ligule color (LgC) 4-5 Ligule shape (LS) 3-4 Colar color (CC) 4-5 Auricle color (AC) 4-5 Culm length (CmL) 5 Culm number (CmN) 6-9 Culm angle (CmA) 7-9 Culm internode color (CmIC) 7-9 Panicle length (PnL) 8 Panicle type (PnT) 8 Secondary branching of panicles (PnBr) 8 Panicle axis (PnAA) 7-9 Awning (An) 7-9 Awn color (AnC) 6 Apiculus color (ApC) 7-9 Stigma color (SgC) 6 Lemma and palea color (LmPC) 9 Lemma and palea pubescence (LmPb) 7-9 Grain length (GrL) 9 Grain width (GrW) 9 Grain shape (GrS) (length-width ratio). 9 Seed coat color (SCC) 9

Primer Test All 34 primers were tested on four diverse rice accessions from different genome groups, to determine the polymorphic primers to use in the study. Amplification of specific products using SSR program (SSR, Simple Sequence Repeat are short monotonous run of sequence most widely used due to its repeatability, reliability and technical simplicity for detection) was carried out. Before this, PCR tubes were prepared and labeled with the four rice

18 accessions. A mastermix was then made for each sample of DNA in 6.6ul reaction volume as follows: MgCl2 (25Mm) 0.5ul Buffer (10x) 1ul dNTP (1Mm) 1ul Taq (5U/ul0 0.1ul Primer “F” (5uM) 0.25ul Primer “R” (5uM) 0.25ul Thereafter 3.6ul of this preparation was pipetted into their respective PCR tubes already containing 3ul DNA. Tubes were tightly closed and mixed on the vortex. The tubes were spun in the centrifuge at 12000 rpm for 30 sec. Amplification of products was checked on 2% agarose gel electrophoresis in 0.5% TBE buffer by loading 5ul of each PCR sample already stained with 4ul loading dye. Out of the 34 primers tested, 14 yielded polymorphism in the four accessions and were subsequently used for fingerprinting.

PCR Amplification PCR reactions were carried out on a PTC-100 Programmable Thermal Controller, MJ Research Inc. The reaction volume was 6.6ul containing 10ng of genomic DNA; 25mM MgCl2; 1mM dNTP mix, 5uM of each Primer, 5 units of Taq polymerase; and 10X Buffer. The temperature cycles were programmed as 940C for 5min followed by 35 cycles of 940C for 1min, 550C for 1min, 720C for 2min, and finally 7min at 720C for final extension. The amplified PCR products were separated in 2% agarose gel electrophoresis and visualized by ethidiun bromide staining. Fragment sizes were estimated from linear interpolination by means of a DNA size standard (1kb plus ladder).

Genetic Similarity and Cluster Analysis Genetic similarity among genotypes was evaluated with a phenetic cluster analysis using the Under-weighted pair grouping with arithmetic average (UPGMA) clustering and a dendogram was derived using NTSYS PC version 2.0 software (Rohlf, 1993).

19 Polymorphism Information Coefficient (PIC) values were calculated for each of the microsatellite loci using the formula developed by Nei (1973):

2 PIC=1-∑x K

Where xk represents the frequency of the kth allele.

Fingerprinting Assessing the level of genetic variation among the cultivated rice varieties or genotypes has been of great importance to taxonomists and is also used in commerce to ascertain the purity of varieties produced. Rice taxonomists are interested in the rapid classification of different taxonomic groups, while breeders are concerned about the determination of usable agronomic variation in breeding programs. In the context of rice cultivars registration, a quick and reliable technique is particularly appealing for cultivars identification. Traditional techniques, based on morphological and cytological characters, breeding behavior and ecological distribution, have been used to assess the genetic variability and relationships among rice cultivars.

The size of the extracted Ofada rice DNA was estimated using the agarose gel electrophoresis; loading buffer, xylene cyanol was added to the DNAs for visualization and loaded into 0.8% agarose gel well (1.2g agarose into 150ml 0.5X TBE buffer). This was allowed to run at 100V for 1 hr. The gel was stained in ethydium Bromide (EtBr – 10mg/ml), and then placed on a light box, while using protective goggle to limit exposure to ultraviolet (UV) light, to view the DNA bands. Finally the gel was photographed using a gel documentation system. The 14 primers were used to fingerprint all the 26 Ofada accessions. Alleles were generated and scored as either present (1) or absent (0) in the accessions. The absence of an amplification product in any of the nine primers in an individual was considered as missing data.

20 3.0 Results 3.1 Physical characterization The physical characteristics are a primary quality in classifying rice grain in rice breeding, marketing, and processing. Variety mixtures were observed (‘Ofada’ 1, 2, 3, 4, 5 and 12 are mixtures of red and white kernel grain rice) (Plates 1-12). Two seed types were observed in Ofada 1, 3 and 5 (Figure 4) and two kernel colors (red and white) in Ofada 12 (Figure 5). Ofada grain size varied significantly from medium or intermediate short to very long grains. Ofada kernel had two distinct colors - red and white. The red kernel types varied from red to deep red. These physical characteristics varied considerably within and between Ofada collections.

Figure 4. Two types of rice hull of Ofada 1 (straw at left, and gold at right)

Figure 5. Two types of rice grain in Ofada 12 (straw hull with red kernel at left and gold hull with white kernel at right)

21 Ofada 1 (Ofada Canada), Ofada 2 (Ofada (Red)

Ofada 3 (Ewekoro 1 Ofada 4 (Ewekoro 2)

Ofada 5 (Ofada) Ofada 6 (Ofada Red

Ofada 7 (Ofada White) Ofada 8 (Ofada Red)

22 1 Ofada 9 (Igbemo Red) Ofada 10 (Igbemo White)

Ofada 11 (Ofada White) Ofada 12 (Ofada)

Plates 1-12. Physical Characteristics of Ofada Rice Seed Samples. 1= Paddy, 2= Brown rice, 3= Milled rice The result (Tables 3a,b) indicates that the grain size is mainly medium to long grain with ‘Ofada’ 8, 9 and 10 having clear long grains. Table 3a: Physical Characteristics of Ofada rice seeds Ofada Length Width L/W 1000 % Hull Hull Kernel Variety (mm) (mm) ratio KW (g) color color Impurity (Shape) (%) 1 6.4 2.8 Medium 24.7 18 Mix Mix 26 2 6.3 3 Medium 25 18.2 Mix Red Pure 3 5.9 3.2 Medium 23.9 19 Gold Mix 22 4 6.9 3 Medium 23.8 20 Gold Mix 14 5 6.8 3.1 Medium 26 16 Mix Mix 18 6 7.2 3.2 Medium 26.8 17.5 Gold White Pure 7 7.6 3 Medium 26.9 17.4 Gold White Pure 8 8 3.1 Long 29 21 Gold White Pure 9 7.6 3 Long 29 21.3 Gold White Pure 10 9 3 Long 31 20.6 Straw White Pure 11 6.8 3.1 Medium 26 20.1 Straw White Pure 12 7 3 Medium 24.9 20.1 Mix Mix 16

The 1000-grain weight also varied among all the entries. Two-hull types were identified-straw-colored and gold or dark colored hull types. With the differences in the weight of hull, fluctuation will be found in the entries thus affecting weight of the brown rice after dehulling. These physical attributes are variety specific and are therefore very important in the classification of any given lot.

23 Similarly the physical characteristics of the 26 Ofada and other rice collections as shown in Table 3b suggest that samples within the Ofada collection contained mixtures (Ofada 1, 3, 5 and 12)

Particle Size Analysis ‘Ofada’ 1-5 has approximately the same particle size (Table 4). ‘Ofada’ 7 was strikingly different from the rest. This result suggests that the ‘Ofada’ lines fall into two variety groups with ‘Ofada’ 7 being a pure bred among the groups.

24 Table 3b. Grain physical characteristics of 26 Ofada and other rice collections

Rice variety Type Length Width Shape Awn Awn color Apiculus Hull Pubescence Kernel (mm) (mm) color color color A 6 3 Medium none _ Straw Straw Smooth Red

Ofada1 B 6.3 3 Medium ‘’ _ Gold Gold Smooth Red

Ofada 2 6 3 Medium ‘’ _ Gold Gold Smooth Red

A 6 3 Medium “ _ Straw Straw Rough Red

Ofada 3 B 6 3 Medium “ _ Straw Gold Smooth Red

Ofada 4 7 3 Long “ _ White Gold Rough Red

A 6.5 2.5 medium “ _ Straw Brown/ Rough Red Ofada 5 Straw B 6 3. 5 medium “ _ Gold Gold Smooth Red

Ofada 6 7 3 long “ Straw Gold Gold Smooth White

Ofada 7 7.5 2.5 long “ _ Gold Gold Smooth White

Ofada 8 8.5 3 Very long “ _ Gold Gold Smooth White

Ofada 9 8 3 Very long “ _ Brown Brown Smooth White

Ofada 10 10 3 Very long “ _ White Straw Smooth White

Ofada 11 7 3 long “ _ White Straw Smooth White

A 7 3 long “ _ Straw Smooth Red

Ofada 12 B 7.5 3 long “ Straw White Gold Smooth White

NERICA 1 7 2.5 long “ - Purple Gold Smooth White

NERICA 3 7 2.5 long _ - Gold Smooth White

NERICA 5 7.5 2.5 long Long Straw Purple Gold Smooth White

NERICA 8 8 2,5 Very long _ _ Gold Gold Smooth White

OS6 7 3.0 long _ _ Straw Straw Smooth White

WAB33-25 7.5 3.0 long _ _ Straw Straw Rough White

WAB189-3B-8-BH 7 3.0 long _ _ White Straw Smooth White

WAB450-1-8-P-160-HB 6 2.5 medium _ _ White Straw/ Smooth White Brown WAB450-24-3-2-P18-HB 7 2.5 long _ _ Straw Brown Smooth Red

WAB706-3-4-K4-KB 8.5 2.5 Very long _ _ Brown Brown Smooth Red

ITA 117 7.5 2.5 long _ _ - Straw Smooth White

ITA 150 8 2.5 Very long _ _ - Gold Smooth White

ITA 301 7.5 2.5 long _ _ - Straw Smooth White

ITA 321 7 3.0 long _ _ - Straw Smooth White

25 Table 4: Ofada Rice Grain size Analysis Compared to known checks

Variety Sieve size/% Retainer 3.35 (mm) 2.336 (mm) 1.70 (mm) 1.18 (mm) 0.85 (mm) OFADA 1 - 68.70 30.90 0.40 - “ 2 - 69.70 30.10 0.20 - “ 3 - 72.90 26.80 0.30 - “ 4 - 62.80 36.10 1.10 - “ 5 - 71.20 28.60 0.20 - “ 6 - 25.90 73.60 0.50 - “ 7 - 10.10 86.80* 2.40 0.70 “ 8 - 31.00 68.40 0.60 - “ 9 - 42.40 55.50 2.10 - “ 10 - 24.60 74.00 1.0 0.40 “ 11 - 43.60 55.90 0.50 - “ 12 - 32.20 67.30 0.50 - ITA 150 - 3.60 90.10* 6.10 0.20 OS 6 - 7.90 83.90* 7.70 0.50 NERICA 1 - 29.00 69.80 0.70 0.50

The results further showed that the ‘Ofada’ rice samples are separated into either medium or long grains. None of the ‘Ofada’ samples or checks had grain particle size of  3.35 mm. ‘Ofada’ 7 and 11 had grain particle sizes as small as 0.85mm along with all the check samples. However, this size percentage was very insignificant. ‘Ofada’ 1, 2, 3, and 4, were medium varieties while ‘Ofada’ 6,7,8,9,10,11,12 and the checks were long grain varieties. The results further showed that ‘Ofada 6,8,10,11 and 12 are more closely related in their particle sizes to the checks (ITA 150) than ‘Ofada’ 1 – 5. ‘Ofada’ 7, which showed exceptional particle size profile, was closely related with the other checks (NERICA 1 and OS6). From the results of this study

26 ‘Ofada’ rice samples can be said to be of two varietal origins with ‘Ofada’ 7 being different in the pack considered.

Milling Characteristics The behavior of rice during milling is one of the most important criteria for rice quality. The milling yield of the 12 ‘Ofada’ entries was generally high with some having over 90% (Table 5). Though total milled rice recovery was high, head rice count was low in most mixtures especially the red-grained samples. The objective of rice milling is to remove the bran layers and germ with minimum breakage of the whole grains, and with the preservation of most of the grain in its approximate original shape. Most red kernel grains have low resistance to milling pressure and therefore break easily and produce high percentage of broken grains. The result showed that the mixed and red kernel grains have low head rice count with high percentage of broken grains. This evidently confirms the earlier assumption that the ‘Ofada’ entries are of two varieties, which might have been mixed on farmers’ fields over the years as variety purity affect these parameters.

Table 5: Milling characteristics of 12 Ofada rice seed samples Ofada 1 2 3 4 5 6 7 8 9 10 11 12 MR 84 91 83 90.8 79.3 91.5 90 94.6 93 96 92 81.4 HR 64 84.2 61.4 86 62.1 86.7 83 87.2 86 88 86.7 60.2 BG 3.2 4.3 3.2 4 6.3 2.8 4.3 2.7 3.1 1.5 2.1 4.2 DG 0.32 0.4 0.45 1.4 2 1.9 2 0.34 0.99 2 2 2.1

MR = Milling Recovery (%), HR = Head Rice (%), BG = % Broken Grain, DG = Damaged Grain (%).

Cooking characteristics Cooking characteristics of rice grain (Table 6) is one of the components of rice quality that largely establishes the economic value and preference of the grain by the end user, especially homemaker. Gelatinization Temperature (GT), a physical property of rice starch, is the range of temperature where at least 90% of the starch granules swell irreversibly in hot water with loss of

27 crystallinity and birefringence. The results showed that the grains were mainly of low (Ofada 1,2,3,4,5,6,7, 11 and 12) to intermediate GT (8,9,10). Some varieties expand more easily in size than others upon cooking; this was considered a highly desirable trait in quality rice. Grain elongation (GE) is a quantitative trait of rice variety. Typically, long grain varieties that cook dry and fluffy are characterized by low to moderate alkali spreading reaction of whole-grain milled rice in dilute alkali; the ‘Ofada’ samples have moderate water uptake ratio and low to moderate gelatinization temperature. As for solids lost in cooking water, most of the rice samples were relatively low, which is a desirable trait. The cooking attributes are comparable with those reported for NERICA 1 and OS6 cultivated in the rice-producing cluster of the Southwest.

Table 6: Cooking characteristics of 12 Ofada rice seed samples

Ofada 1 2 3 4 5 6 7 8 9 10 11 12 GT L L L L L L L I I I L L WUR 43 39 41 46 38 40 47 46 42 43 41 43 GE 75 81 79 63 69 73 71 66 68 70 69 72 SCW 1.2 0.9 2.1 1.4 1.7 1.1 1.0 0.8 0.8 1.1 1.4 1.5 CT 17 18 22 21 18 19 24 22 22 21 22 23

GT=Gelatinization Temperature, WUR=Water Uptake Ratio (%), GE=Grain Elongation during cooking (%), SCW=Solids in Cooking Water (%), CT=Cooking time (min), L=low GT, I=intermediate GT.

28 Pasting Characteristics

Table 7: Pasting characteristics of 12 Ofada rice seed samples and checks Ofada PV TV BV FV SV PT PaT 1 124.83 113.08 11.75 226.00 112.92 6.20 64.95 2 121.58 109.17 12.42 217.42 108.25 5.93 64.95 3 115.00 110.58 4.42 224.25 113.67 6.40 64.95 4 125.17 113.08 12.08 208.92 104.85 6.27 64.95 5 138.50 116.50 22.00 226.08 109.58 5.67 64.95 6 123.50 109.83 13.67 223.42 113.58 5.93 64.95 7 125.67 101.50 6.92 232.75 131.25 8.99 64.30 8 112.92 80.50 16.75 215.92 135.42 8.99 64.50 9 152.42 117.50 34.92 261.17 143.67 5.12 64.00 10 123.83 105.83 13.25 229.17 123.33 8.99 64.35 11 117.17 92.42 10.17 228.00 135.58 8.99 64.50 12 117.17 81.75 11.58 221.17 139.42 8.99 64.00 ITA 150 121.60 114.20 7.40 273.10 158.90 6.30 64.50 OS 6 89.30 71.30 18.10 229.30 149.80 7.0 64.50 PV = Peak Viscosity, TV = Trough Viscosity, BV = Break down Viscosity, SV = Setback Viscosity, PT = Peak time, PaT = Pasting Temperature.

The most important property of starch, the major component of rice is its ability to swell and produce a viscous paste when heated in water. As the temperature further increases, the granules rupture and this permits further hydration and irreversible granule swelling, a process termed gelatinization. Granule rupture and subsequent polymer alignment, due to the mechanical shear, reduces the apparent viscosity of the paste. These combined processes that follow gelatinization are termed pasting. The pasting characteristics of ‘Ofada’ rice samples with checks are shown in Table 8. Critical observations of the results indicated that the samples had similar values in almost all pasting parameters. Any observed variations were more due to particle size differences of the raw samples than from varietal difference. All the varieties were pasted at 64  0.5oC reached peak viscosity within 9 minutes. The results showed that the minimum temperature required to cook the ‘Ofada’ samples is 64  1oC. The viscogram (Appendix 3) showed that the

29 ‘Ofada’ samples and checks exhibited non-waxy cereal starch pasting characteristics as expected. The ‘Ofada’ samples had similar pasting characteristics with the checks. As expected, all the samples showed high peak and set back viscosities indicating that, like all other cereal starches, the starch consistency will be low and subject to retro gradation on standing. Their noticeable trough is an indication that the native starch suffered little or no damage during processing. The final viscosity indicated that like all other cereal starch, the viscosity of the paste will increase on cooling and is an indicative of high amylase contents.

Chemical characteristics The protein content was generally uniform, and this may be attributed to cultural practice during the production of rice on the field e.g. rate of nitrogen fertilizer application.

Table 8: Proximate composition of ‘Ofada’ rice seed samples

Ofada rice samples Protein (%) Ash(%) Moisture(%) 1 5.25 0.5 12.6 2 5.65 0.5 13.4 3 5.70 0.5 13.8 4 6.57 1.0 13.6 5 7.50 1.0 12.9 6 5.43 0.6 11.8 7 7.06 1.0 13.6 8 6.44 1.2 13.1 9 6.75 0.5 13.2 10 6.81 0.5 13.2 11 7.88 1.0 12.6 12 7.58 0.5 12.8 Checks ITA 150 6.31 0.12 11.4 OS6 6.63 0.13 114

Amino Acid Profile (AAP) The amino acid profile (Table 8) showed that all the ‘Ofada’ samples including the checks had high lysine content (>5.0g/100g), which is usual for cereals. Strikingly, some of the ‘Ofada’ samples showed high levels of methonine especially Ofada 11 (5.26g/100g) and ‘Ofada’ 8 (4.08g/100g) while ‘Ofada’ 2,3, 4 and 12 had methionine contents of  4.0g/100g). The results were

30 generally indicative that the ‘Ofada’ samples were similar in their AAP. There were also no significant (<0.05) differences in the AAP of the ‘Ofada’ samples compared to the checks. Amino acid profile is basically a genetic factor issue and could be affected by soil type and profile and other agronomic conditions. From the results obtained, the AAP of the Ofada varieties and the checks suggests that the samples are of similar genetic origin. There were no striking differences in the peaks recorded in the chromatographs (Appendix 4). The high lysine and methionine content of most of the ‘Ofada’ samples, it is suggests that these factors should be investigated a bit further.

Morphological characterization There was considerable morphological variability among Ofada rice collections from farmers’ fields. NERICAs are shorter than the Ofada rices. However, three WAB accessions (WAB189-B-B-B-8-HB, WAB450-1-B-P-160-HB and WAB450-24-3-2-P18-H) showed seedling heights that were comparable with those of the Ofadas. Moreover, ITA varieties, especially ITA117, a typical upland rice variety, are shorter probably because of the increased water regime (Table 9).

As shown in Table 9 and Appendix 2, morphological variability between and within Ofada rices was also noticeable. There was disparity of plant height, grain types, flag leaf angles and flowering (Figures 6 and 7). Generally, Ofada 1, 2, 3, 4, 5, 6 and 12 initially looked similar to OS6, while Ofada 7, 8 and 9 appeared to be the same based on plant height and flowering time. Data collected were just the average but the standard deviations were high within the Ofadas. The analyzed data show the specific variations/ mixtures within Ofada rices compared to OS 6, ITA 150 and NERICA 1. Because of the high variability observed within the Ofada collections, there is no obvious similarity between them and OS6, ITA50 and NERICA1. Even rouging out the obvious mixtures cannot determine the real Ofada rice unless specific criteria are available.

31 Figure 6. Different grain types within Ofada 10.

Figure 7. Different rice types, plant height and flowering within Ofada 5

32 Table 9: Agronomic characteristics of the 12 Ofada rice seeds from farmers, NERICAS and ITA rice under lowland condition in 2007 dry season at Ibadan

Plant height Plant Tiller Panicle Maturity (cm) @ 5-leaf height (cm) count count/stand (days) Varieties stage @ maturity 50% flw Ofada 1 67.0 114.0 9.7 6.5 71 101 Ofada 2 57.5 101.4 7.4 5.6 69 99 Ofada 3 65.5 109.7 7.2 4.7 68 98 Ofada 4 57.0 107.2 6.6 4.5 70 100 Ofada 5 63.5 102.2 7.2 6.4 65 95 Ofada 6 65.0 111.2 6.3 5.0 62 92 Ofada 7 65.5 95.7 5.5 4.0 50 80 Ofada 8 58.5 102.2 7.8 5.3 53 83 Ofada 9 61.0 95.1 6.4 4.2 52 82 Ofada 10 63.0 104.6 5.0 5.0 57 87 Ofada 11 67.5 105.8 7.3 6.0 55 85 Ofada 12 58.5 112.4 8.1 5.0 69 99 NERICA 1 54.5 85.1 8.5 5.6 51 81 NERICA 3 49.5 83.0 8.1 4.4 67 97 NERICA 5 54.5 85.1 9 6 63 93 NERICA 8 50.0 83.0 13 6 64 94 OS6 58.0 107.8 6.5 7 63 93 WAB33-25 67.5 99.6 7 5 55 85 WAB189-B-B-B-8-HB 62.5 90.5 5.5 3.8 57 87 WAB450-1-B-P-160-HB 62.5 90.5 7 4 65 95 WAB450-24-3-2-P18-HB 64.5 103.5 10 6 62 92 WAB706-3-4-K4-HB 56.5 86.3 8 6 51 81 ITA117 34.3 82.5 21 6 ITA150 55.5 103.5 6.9 3.8 62 92

33 Molecular Characterization Fingerprinting Genotypic scores were generated and visual evaluation indicates that all the 14 SSR primers used produced 203 readable bands and demonstrated genetic variability among Ofada rice accessions, NERICAs and ITA varieties. The gel pictures of Ofada and NERICA genotypes are shown in Figures 8 and 9.

1 2 3 4 5 6 7 8 9 10 11 12L10L50L100

Figure 8. Twelve Ofada lines from PrOpCom 1-12 are the Ofada1-12 accessions, L10: 10ng/ul Lambda DNA, L50; 50ng/ul Lambda DNA, L100: 100ng/ul Lambda DNA

L10 L50 L100 N1 N3 N5 N8 OS6 13 14 15 16 17 18 19 20 21

Figure 9 Genomic DNA of the NERICA/WARDA/ITA varieties L10: 10ng/ul Lambda DNA, L50; 50ng/ul Lambda DNA, L100: 100ng/ul Lambda DNA, N1- Nerica 1, N3-Nerica 3, N5-Nerica5, N8-Nerica8,13-WAB33-25, 14-WAB189-B-B-B-8-HB, 15- WAB450-1-B-P-180-HB, 16-WAB450-24-3-2-P18-HB,17-WAB706-3-4-K4-KB,18-ITA117, 19- ITA150, 20-ITA301, 21-ITA321 35

Polymorphism of microsatellite markers The fourteen primer pairs used in the study generated polymorphic bands among the genotypes. A total of 41 alleles were detected among the 26 rice genotypes with an average of 2.9 alleles per locus. The PIC values for the microsatellite loci ranged from 0.2112 to 0.645 with an average of 0.46. The low PIC values were observed for the primers of RM3 (0.231), RM115 (0.211), RM124 (0.237) and RM185 (0.237). The PIC values for the remaining microsatellite loci were all above 0.4 (Table 10).

Theoretically, PIC values can range from 0 to 1. At a PIC of 0, the marker has only one allele. At a PIC of 1, the marker would have an infinite number of alleles. A PIC value greater than 0.7 is considered to be highly informative, whereas a value of 0.4 is considered to be moderately informative (Hildebrand et al., 1992). Table 10. Allele variation and PIC value for microsatellite loci (SSR) identified in 26 genotypes SSR locus Total allele PIC values RM3 3 0.231111 RM9 3 0.645136 RM18 2 0.535503 RM115 3 0.2112 RM124 3 0.237812 RM131 3 0.62585 RM184 3 0.470868 RM185 3 0.237812 RM188 3 0.624444 RM191 3 0.482422 RM214 3 0.474506 RM223 3 0.607422 RM225 3 0.495317 RM282 3 0.565158 Mean 2.9 0.46

35 36

Cluster analysis of DNA polymorphism The genetic relationships among the rice genotypes are presented in a dendogram based on the informative microsatellite alleles (Figure 10). The entire rice genotypes grouped into two main branches in the dendogram at 0.51 coefficient – Ofada 1-5 (group 1), and all the others (group 2). However, at a coefficient of 0.62, group 2 is further subdivided into two clusters - Ofada 6-10, WAB189-3B-8-HB, ITA 321, WAB450-1-8-P-160-HB, OS6 and WAB33-25 (group 2A) and Ofada 11 and 12, WAB450-24-3-2-P18-HB, WAB706-3-4-K4-KB, ITA117 and ITA301 (group 2B). Ofada 6-10 are therefore more closely related to each other than they are to Ofada 11 and 12.

Figure. 10. Cluster of 26 rice genotypes based on non-weighted pair grouping with arithmetic average (UPGMA) clustering and plotted in a phenogram using NTSYS-PC version 2.0J

Cluster analysis of genetic distances showed a distant relationship within the Ofada 7,8,9 and10 and OS6, ITA150 and NERICA1, while Ofada 11 and 12 are only distantly related to this group and this explains the differences in the time to maturity.

36 37

Summary of Findings 1 There were apparent mixtures in the color of the rice grain in terms of hull and kernel color with straw and gold colors being predominant. Grain kernels were mainly red and white. The red kernels suggest contamination from Oryza glaberrima Steud (indigenous Africa Rice). 2 There was significant variation in the grain size of Ofada rice, which ranged from medium or intermediate to very long grains. The kernels were red and white, the red kernel types varying from red to deep red. 3 The entire samples showed high milling returns, but head rice count showed that all the mixed grains with O. glaberrima seeds had low head rice count. 4 Gelatinization test shows two distinct categories, low and intermediate categories. But the water uptake was uniform for all the samples. There were no differences observed in cooking time. 5 The peak viscosity, breakdown viscosity, peak time and pasting temperature were uniform for all the varieties, including the checks. 6 Proximate composition and amino acid profile showed similar values for the Ofada rice varieties and the checks. 7 Based on the dendogram the following comparisons can be made: OS 6 and WAB33-25 are the same and are in the same group as Ofada 10, WAB450-1-8-P-160-HB, WAB189-3B-8-HB and ITA321; ITA150 is close to Ofada 6,7,8 and 9, NERICAs 1, 3, 5 and 8; Ofada 1-5 belong to one group distinct from all the other varieties; while Ofada 11 and 12 are closely related to WAB450-24-3-2-P18-HB, WAB706-3-4-K4-KB, ITA117 and ITA301. 8 For seed purification, 10 individual plants per Ofada collection per replication were harvested. These plant sections will be planted side by side in the 2007 rainy season. Based on specific criteria (grain color, size and shape, height, etc.), the rows matching the Ofada characteristics will be selected for seed multiplication and further morphological

37 38

characterization and molecular profiling to assure purity and establish proper identity. 9 The variability within and between samples explains the complexity in identifying the real Ofada. 10 The information generated from the molecular characterization would have been more reliable if the seed was not mixed. Mixed seeds within Ofada accession (collected from farmers) will give a mixed DNA, which is not a good material for DNA fingerprinting. Molecular characterization must be done after the Ofada rice accessions have been purified and isolated.

Recommendations on further work to address the problem of seed mixtures and adulteration The following actions are recommended for purifying and identifying the true Ofada rice: 1. Separate and purify the obvious lowland types in the Ofada collections. 2. Conduct harvesting of all rice types within the Ofada rice (1-12). 3. Plant one panicle in a row and compare with OS 6, ITA150 and NERICA1 for morphological characterization – this should provide very useful information on the real Ofada. 4. Discard the obvious mixtures and harvest rows that are not segregating (from the panicle to row nursery). 5. Conduct the molecular fingerprinting of the harvested pure types. 6. Multiply seeds of the harvested Ofada types for grain quality analysis. 7. Subject the pure lines to stakeholder (producers, marketers, consumers) analysis and identify the best Ofada variety. 8. The consumer study report indicates that both white and red-grained Ofada rice are acceptable to consumers, although the red is regarded as the real Ofada rice. Consequently, it may be appropriate to do the following (please refer to Table 2 and Figure 10):

38 39

 Purify Ofada 1-5 and 12 for farmers in Ogun and Lagos States.  Purify Ofada 6-11 and 12 for farmers in Osun, Ekiti and Lagos States. This process will take another two cropping seasons (wet and dry) to purify and fully characterize the Ofada rices.

Conclusions Grain quality characteristics are very difficult to define with precision as preferences vary among consumers, processors, homemakers, researchers etc. Variety quality traits therefore, may be considered from the viewpoint of physical (grain size, shape, appearance and uniformity), milling and cooking characteristics. The grains were broadly grouped into two-variety types based on the hull and kernel color. Grain physical dimensions also classified the grains into medium to long grain types and this was substantiated by the sieve analysis. However, research has shown that even within the rice plant there might be differences in grain physical characteristics. There was a high milling return, but the suspected impurities gave rise to the low head rice count. This suggests that the original variety was contaminated by the local rice varieties over the years. The results for the cooking characteristics showed that the samples had high peak and setback viscosities, which were expected. It indicated that, like all other cereals, starch consistency would be low and subject to retro gradation on standing. The high peak viscosity was indicative of high amylose content. The Ofada rice samples related closely to the checks in all these parameters based on physical and chemical analysis. Differences were due to particle size differences, not varietal differences. Results of this study showed that the ‘Ofada’ rice varieties have similar amino acid profiles and related closely to two of the checks (ITA 150 and OS 6). From the physical and chemical analysis, it is concluded that ‘Ofada’ rice is one variety that has been contaminated over the years with other varieties of rice from the farmers’ fields. The differences in grain hull and kernel weight, particle sizes etc arose from this contamination and not from genetic differences. Ofada rice as observed by consumers (CMRG Ltd, 2006) are

39 40 short, bold, and has brown stripes and high swelling capacity with good aroma. These observations are substantiated by the grain physical characteristics, as most of the grains are bold and medium shaped. The aroma may be subject to further investigation, as the sauce used in eating Ofada rice is prepared with locust beans. The stripes are as result of partial milling of the brown or red kernel grains. The grains also swell above 60% and attract high command of patronage from the consumers. It is evident from molecular characterization that Ofada 1-5 belong to one group, Ofada 6-10 belong to another group while Ofada 11-12 fall in completely different group of their own. Thus, from this study, it can be safely concluded that Ofada 1-5 from Ogun State show different characteristics from those from Osun, Ekiti and Lagos States, as indicated by the 1000 grain weight, percent hull, particle size analysis and milling characteristics and molecular characterization. It will therefore be necessary to purify the ofada collections to address the issue of adulteration; seed mixtures and the resultant low yield on farmers’ fields as follows: purify Ofada 1-5 and 12 for farmers in Ogun and Lagos States. Purify Ofada 6-11 and 12 for farmers in Osun, Ekiti and Lagos States. Also source for fresh foundation seeds of ITA 150 (FARO 46) and OS 6 (FARO 11) already released as varieties in Nigeria from National Seed Service (NSS) in collaboration with NCRI and distributed to farmers for cultivation in the Northwest rice growing clusters in Nigeria.

40 41

References

CMDG Ltd (Communication and Marketing Research Group Limited) (2006). Draft Report on Project Delicacy (Ofada Rice Attributes Evaluation Study) submitted to PrOpCom, Abuja.

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Garris, A.J., T.H. Tai,J. Coburn, and S. McCouch, (2005). Genetic structure and diversity in Oryza sativa L. Genetics 169: 1631-1638.

Hildebrand, C.E., D.C. Torney and R.P. Wagner, (1992). Informativeness of polymorphic DNA Markers. Los Alumnus Science, Number 20, 1992.

IRRI (1996). Standard Evaluation Scales for rice. Los Banos , The Phillippines

Nei, M., (1973). Analysis of gene diversity in subdivided populations, Proc. Natl. Acad. Sci. USA 70: 3321-3323.

Ren, F., B.R.Lu, J.Huang, and Y.Zhu, (2003). A comparative study of genetic relationships among the AA genome Oryza species using RAPD and SSR markers. Theor Appl Genet 108: 113-120.

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Virk, P.S., J.Zhu, H.J. Newbury, G.J. Bryan, M.T.Jackson, and B.V. Ford-Lyod, (2000). Effectiveness of different classes of molecular marker for classifying and revealing variation in rice (Oryza sativa) germplasm. Euphytica,112: 275-284.

41 Appendix 1, Amino Acid Profile of ‘Ofada’ rice Samples (12) and Checks (g/100g Protein)

Amino 1 2 3 4 5 6 7 8 9 10 11 12 ITA OS6 N1 Acids 150 Lysine 4.20 4.70 4.88 5.50 5.00 4.80 5.00 5.87 4.24 4.50 5.60 5.18 4.02 4.10 4.02 Histidine 2.40 3.00 3.68 3.63 3.00 3.00 2.99 3.75 2.49 2.27 3.51 3.28 2.08 2.70 3.20 Arginine 5.30 5.94 6.60 7.03 7.00 5.64 5.69 6.84 5.51 5.97 6.39 6.52 5.00 5.21 6.00 Aspartic Acid 6.90 7.00 7.11 7.02 6.90 7.00 6.89 6.96 6.96 7.02 6.60 6.81 6.50 7.10 7.60 Threonine 2.35 3.00 3.35 3.49 3.08 3.50 3.29 3.70 2.51 2.23 3.25 4.03 3.01 2.90 3.41 Serine 3.91 4.86 4.77 5.29 5.00 4.70 4.59 5.46 3.79 3.98 4.95 4.98 3.80 4.00 3.80 Glutamic acid 10.80 12.94 13.47 12.91 12.80 13.30 13.05 12.96 12.00 11.27 13.84 12.96 10.96 12.00 11.71 Proline 2.25 3.06 2.95 3.40 3.22 2.40 2.46 3.59 2.35 2.31 2.82 2.95 2.04 3.00 2.50 Gycine 3.01 2.70 2.89 3.08 3.00 2.80 2.84 3.00 2.99 2.89 2.89 2.74 3.50 2.60 3.70 Alanine 3.11 3.74 3.80 3.94 3.94 3.90 3.99 4.19 2.98 3.20 3.87 3.89 3.86 2.60 4.02 Cystine 1.05 1.30 1.37 1.29 1.45 1.25 1.18 1.53 1.00 1.04 1.44 1.29 1.17 1.22 0.92 Valine 4.06 5.50 6.00 5.94 5.47 4.90 4.99 5.97 4.50 4.51 5.03 5.58 4.50 4.01 4.40 Methionine 2.06 3.64 3.89 3.95 3.19 3.36 3.49 4.08 2.22 2.15 5.26 3.56 2.91 2.03 2.21 Isoleucine 4.03 4.03 4.89 4.99 4.31 4.76 4.79 5.10 3.24 3.89 5.03 4.69 3.99 3.00 4.04 Leucine 5.50 6.00 6.18 6.47 5.95 5.19 5.64 6.57 4.98 5.29 5.50 6.01 5.21 5.01 6.01 Tyrosine 3.73 5.53 4.90 5.53 5.67 4.15 4.70 5.67 5.58 3.87 4.70 5.00 3.73 4.15 4.32 Phenylalanine 3.81 4.02 4.26 4.50 4.10 4.24 4.31 4.58 3.94 3.86 4.65 4.26 4.40 4.00 4.07 43

Appendix 2. Morphological characteristics of the 12 Ofada rice from farmers, NERICAS and ITA rice under lowland condition in 2007 dry season. LgL Varieties LL (cm) LW (cm) LBP LBC BLSC LA FLA (mm) LgC LS AC 50% Flw. Ofada 1 41.0 1.4 Pbsc Gr Gr. H H 5 Wh Cft Lgr 71 Ofada 2 53.0 1.3 Int Gr. L.Ppl H Dsc 19 Wh Cft Lgr 69 Ofada 3 40.0 1.4 Pbsc Gr. Gr. H E 9 Wh Cft Lgr 68 Ofada 4 44.0 1.2 Int. Gr. Gr. H Dsc 10 Wh Cft Lgr 70 Ofada 5 38.0 1.3 Pbsc. L.gr. L.Ppl E E 12 Ppl Cft Lgr 65 Ofada 6 40.0 1.3 Glbr. Gr Gr. H E 14 Ppl Cft Lppl 62 Ofada 7 33.0 1.2 Glbr. Gr. Ppl H Int 12 Ppl Cft Ppl 50 Ofada 8 34.0 1.2 Glbr. Gr. Gr. H Int 10 Ppl Cft Lgr 53 Ofada 9 33.0 1.3 Int. D.Gr Ppl E E 10 Ppl Cft Lgr 52 Ofada 10 37.0 1.3 Glbr. Gr L.Ppl E Int 10 Ppl Trc Ppl 57 Ofada 11 39.0 1.4 Pbsc L.gr. Gr. Dr H 12 Ppl Cft Ppl 55 Ofada 12 38.0 1.3 Glbr. Gr. Gr. H E 5 Wh Cft Lgr 67 NERICA 1 36.0 1.3 Pbsc. D.gr. Lppl E Int 3 Ppl Cft Gr 51 NERICA 3 39.3 1.8 Pbsc Gr. Gr. Dr Int 5 Ppl Cft Gr 67 NERICA 5 39.2 1.3 Pbsc Gr. Ppl. E E 2 Ppl Cft Gr 63 NERICA 8 37.3 2.3 Pbsc Gr. Gr. Dr Int 3 Wh Cft Gr 64 OS6 38.2 1.4 Pbsc L.gr. Ppl. Dr Int 12 Wh Cft Lgr 63 WAB33-25 31.3 1.4 Pbsc Gr. Gr. E E 9 Wh Trc Lgr 55 WAB189-B-B-B-8-HB 33.9 1.4 Int. L.gr. Gr. H Dsc 10 Wh Cft Gr 57 WAB450-1-B-P-160-HB 34.4 1.3 Pbsc Gr. Gr. E Int 6 Ppl Cft Gr 65 WAB450-24-3-2-P18-HB 37.7 1.2 Pbsc L.gr. Gr. Dr Dsc 3 Ppl Cft Lgr 62 WAB706-3-4-K4-HB 28.9 1.3 Pbsc Gr. Gr. Dr Dsc 9 Ppl Cft Gr 51 ITA117 40.3 1.1 Pbsc Gr. Lppl E Dsc 10 Ppl Cft Gr 62 ITA150 39.2 1.2 Pbsc Gr Gr. E Int 5 Ppl Cft Lgr 64 Leaf length; LW = Leaf width; LBP = Leaf pubescence; LBC = Leaf blade color; BLSC = Basal leaf blade color; LA = Leave angle; FLA = Flag leaf angle; LgL = Ligule length; LgC = Ligule color; LS = Ligule shape; AC = Auricle color; Ppl = Purple; Lppl = Light purple; Dppl = Dark purple; Dgr = Dark green; Ac-acm = Acute to acuminate; Cft = Cleft; CmL = Culm length; CmN /TilN = Culm number /Tiller number; CmA = Culm angle; PnL = Panicle length; PnT = Panicle type; LmPC = Lemma and palea color ; LmPb = Lemma and palea color pubescence; Pbs = Pubescent ; Glbr = Glabrous; Dsc = Descending; E = Erect; H = Horizontal; Wh = White; Trc = Truncate.

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Appendix 3 Viscograms of Ofada Pasting Characteristics 240 240 Final = 223.42 Final = 217.42

180 180 U U V V R R

y y

t Peak = 123.50 t Peak = 121.58 i 120 i 120 s s

o Hold = 109.83 o Hold = 109.17 c c s s i i V V

60 60

0 Newport Scientific Pty Ltd 0 Newport Scientific Pty Ltd 00 3 6 9 12 1515 00 3 6 9 12 1515 Time mins Time mins OFADAOFA 1a 1 OFADAOFA 2a2

240 240 Final = 224.58 Final = 208.92

180 180 U U V V R R

y y Peak = 125.17 t t

i 120 Peak = 114.58 i 120 s s o o Hold = 113.08

c Hold = 108.00 c s s i i V V

60 60

0 Newport Scientific Pty Ltd 0 Newport Scientific Pty Ltd 00 3 6 9 12 1515 00 3 6 9 12 1515 Time mins Time mins OFA 3a OFA 4b OFADA 3 OFADA 4

240 Final = 246.25 Final = 226.08 240

180

U 180 V U R Peak = 138.50 V

R y

t i 120 y s t i

o Hold = 116.50 120 s c o s Peak = 97.42 i c s V i Hold = 86.42 60 V 60

0 Newport Scientific Pty Ltd 00 3 6 9 12 1515 0 Newport Scientific Pty Ltd Time mins 00 3 6 9 12 1515 OFA 5a Time mins OFADA 5 OFA 6a OFADA 6

Final = 246.33 240 240 Final = 234.42

180

180 U U

V Peak = 150.50 V R

R

y t y Hold = 132.42 i t 120 s i 120 s o o c c s i s Peak = 88.17 i V V Hold = 76.58 60 60

0 Newport Scientific Pty Ltd 0 Newport Scientific Pty Ltd 00 3 6 9 12 1515 00 3 6 9 12 1515 Time mins Time mins OF 6 OFA 4 b OFADA 7 OFADA 8

240 240 Final = 224.25 Final = 226.00

180 180 U U V V R R

y y Peak = 124.83 t t i 120 Peak = 115.00 i 120 s s o Hold = 110.58 o Hold = 113.08 c c s s i i V V

60 60

0 Newport Scientific Pty Ltd 0 Newport Scientific Pty Ltd 00 3 6 9 12 1515 00 3 6 9 12 1515 Time mins Time mins OFA 3 b OFA 1b OFADA 9 OFADA 10

240 240 Final = 224.83 Final = 215.42

180 180 U U V V Peak = 143.25 R R y y t t i 120 Peak = 117.33 i 120 s s Hold = 120.08 o o

c Hold = 108.33 c s s i i V V

60 60

0 Newport Scientific Pty Ltd 0 Newport Scientific Pty Ltd 00 3 6 9 12 1515 00 3 6 9 12 1515 Time mins Time mins OFADAOFA 2 11b OFADAOFA 12 5b 48

Appendix 4. Chromatograms of Ofada Rice Seed Samples with Hecks

Ofada 2 Ofada 1

Ofada 3 Ofada 4

Ofada 5 Ofada 6

Ofada 7 Ofada 8

48 49

Ofada 9 Ofada 10

Ofada 11 Ofada 12

0S6 ITA150

49